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Benzyl and Benzaldehyde-Derived Metabolites from Desert Actinomycetes Sp. CDS1.

Byline: Mamona Nazir, Rizwana Mustafa, Muhammad Imran Tousif, Shabir Ahmad, Tasneem Khatoon and Ishtiaq Ahmad

Summary: Chromatographic purification of the culture broth of Actinomycetes CDS1 provided five new (1-5) and three known (6-8) metabolites. The new compounds 1, 4 and 5 were elucidated due to 1D-, 2D-NMR techniques, FABMS and HR-FABMS analyses. The nature of glycosidic part in 4 was confirmed through optical rotation value and comparison of the Rf value of hydrolyzed sugar with that of standard sample. Compounds 2 and 3 were tentatively identified due to 1H NMR and +ve FAB-MS analysis as mixture in one of the polar minor fractions. Structures of the known compounds 6-8 were determined due to 1H NMR and FABMS analysis.

Keywords: Desert soil, Actinomycetes, Secondary metabolites, Structure elucidation.

Introduction

Among numerous classes of microorganisms, Actinomycetes are the bacteria which showed chemical and morphological diversity. They are widely distributed in terrestrial and marine environment and constitute a potential source of novel-small-to-macromolecules, with wide range of bioactivities. The main classes of compounds produced by Actinomycetes include polyene, anthracyclines, aminoglycosides, streptothricins, cyclopolylactones, actinomycins, polyether and quinoxaline-peptides, [1] however, an Actinomycetes sp. CDS1, isolated from the sandy soil of Cholistan Desert was cultured in laboratory. The ethyl acetate extract of the fermentation broth provided benzyl or benzaldehyde based several small molecules. The present paper describes the isolation and structure elucidation of five new (1-5) and three known compounds 6, 7 [2] and 8 [3] (Figure 1) from the culture broth of Actinomycetes sp. CDS1.

Experimental

General experimental procedures

Optical rotation values were measured on a JASCO DIP-360 polarimeter. UV spectra were obtained in methanol on U-200 Schimadzu UV-240 spectrophotometer. Infrared (IR) spectra were recorded as KBr pallets on Shimadzu 460 spectrometer. The 1H and 2D NMR spectra were recorded on a Bruker AM-300 MHz spectrometer; whereas, 13C NMR data were recorded on the same machine operating at 75 MHz. All the spectra were recorded in deuterated solvents, while the chemical shift values (I') are reported in ppm and the coupling constant (J) are in Hz. Chromatographic separations were carried out using aluminium sheets pre-coated with silica gel 60 F254 (20x20 cm, 0.2 mm thick; E. Merck) for thin layer chromatography (TLC) and silica gel (230-400 mesh) for column chromatography. TLC plates were visualized under UV at 254 and 366 nm and by spraying with ceric sulphate reagent solution followed by heating.

RP-18 silica gel was used for VLC where various compositions of aqueous acetonitrile were used as mobile phase. Sephadex LH-20 was also used to clean the separated compounds or fractions. Authentic sample of L-Rhamnose was purchased from Sigma-Aldrich 83650, through local supplier. ESIMS and HRESIMS were recorded on a micrOTOF time of flight mass spectrometer (Bruker Daltonics, Bremen, Germany), as well as on an Apex IV 7T Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Billerica, MA). FABMS and HRFABMS were recorded on Finnigan (Varian MAT) JMS-HX 110 with a data system and JMSA 500 mass spectrometers, respectively.

Isolation of Actinomycetes CDS1

The bacterium was isolated from the sandy soil of Cholistan Desert on chitin agar supplemented with cycloheximide, following the known procedures published by S. K. Agadagba [4].

Cultivation of bacteria and extraction

20 L of the culture medium was prepared as: 200 g malt extract, 80.0 g yeast extract and 80 g glucose dissolved in 20 L of tap water and pH was adjusted to 7.8. The medium was then transferred to 1.0 L Erlenmeyer flasks (250 ml each), and was autoclaved. All the flasks were inoculated under sterilized conditions with pieces of an agar culture of Actinomycetes sp. CDS1. The flasks were then incubated on a linear shaker (120 rpm) at 28 AdegC for 14 days, until the broth became dark brown in color. The culture broth was harvested after 14 days, mixed with celite, and filtered off under pressure. The filtrate was extracted with EtOAc to get dark brown extract (2.3 g).

Purification and characterization

The ethyl acetate extract of the culture broth was subjected to silica gel column chromatography using a gradient (100:0 to 90:10) of CHCl3/MeOH. As a result four fractions (S1-S4) were collected based on their TLC profiles. Fraction S2 was subjected to RP-18 VLC using 30% aqueous acetonitrile to get compound 1 (7.5 mg), 6 (4.0 mg) and 8 (5.8 mg), along with another impure fraction, which on passing through Sephadex LH-20 with methanol gave compound 7 (8 mg). Main fraction S4 was also subjected to RP-18 VLC using gradient of water:acetonitrile (80:20 to 0:100) to get three daughter fractions (S4a-S4c). Fraction S4a and S4c showed one spot each on TLC with minor impurities and thus were passed separately through Sephadex LH-20 column with methanol to get compound 4 (8.2 mg) and 5 (5.3 mg) respectively. Fraction S4b displayed two major compounds on TLC with minor impurities; however, due to its low amount (max (MeOH) nm (log Iu): 245 (4.10), 289 (3.58); (-)-ESIMS: m/z 180 [M-H]-; (-)-HR-ESIMS: m/z 180.0658 [M-H]-calcd. 180.06553 for C9H10NO3 corresponding to the actual formula as C9H11NO3.

N-[2-O-[alpha]-L-rhamnosyl-5-(hydroxymethyl)phenyl] acetamide (2) and N-(5-formyl-2-O-[alpha]-L-rhamnosylphenyl) acetamide (3)

White amorphous solid (< 2mg); 1H NMR (CD3OD, 300 MHz): I' 9.83 (s), 7.64, 7.65 (d, J = 1.7Hz), 7.03, 7.00 (dd, J = 8.3, 1.6 Hz), 6.84, 6.83 (d, J = 8.6 Hz), 5.43, 5.40 (d, J = 1.4 Hz), 4.50 (s), 4.35-3.41 (several overlapped signals of rhamnose), 2.18 (s), 1.25, 1.23 (d, J = 6.1 Hz); +HR-FABMS: m/z 350.1214 [M+Na]+ (calcd. 350.1216 for C15H21NNaO7) and 348.1053 [M+Na]+ (calcd. 348.1059 for C15H19NNaO7).

2,4-Dihydroxy-3-O-[alpha]-L-rhamnopyranosyl-benzaldehyde (4)

White amorphous powder (8.2 mg); IR (KBr): 3410, 2905, 2800, 1640, 1510, 1490 cm-1; 1H NMR (CD3OD, 300 MHz): I' 9.86 (1H, s, H-7), 7.31 (1H, d, J = 8.6 Hz, H-6), 6.56 (1H, d, J = 8.6 Hz, H-5), 5.40 (1H, d, J = 1.6 Hz, H-1'), 4.33 (1H, dq, J = 9.7 and 6.2 Hz, H5'), 4.17 (1H, dd, J = 3.3 and 1.7Hz, H-2'), 3.93 (1H, dd, J = 9.5 and 3.4 Hz, H-3'), 3.43 (1H, t, J = 9.6 Hz, H-4'), 1.21 (3H, d, J = 6.2 Hz, H-6'); 13C NMR (125 MHz, CD3OD): 196.4 (CH-7), 159.9 (Cq-4), 157.8 (Cq-2), 132.6 (Cq-3), 131.9 (CH-6), 116.7 (Cq-1), 110.0 (CH-5), 102.9 (CH-1'), 73.7 (CH-4'), 72.1 (CH-3'), 72.0 (CH-2'), 71.2 (CH-5'), 17.9 (CH3-6'); (+)-FABMS: m/z 323 [M+Na]+, 176 [M+Na-sugar]+; (+)-HR-FABMS: m/z 323.07394 [M+Na]+ (calcd 323.07364 for C13H16O8Na, corresponding to the formula as C13H16O8).

2, 4-Dihydroxy-3-O-[2-OMe]-[alpha]-L-rhamnopyranosyl-benzaldehyde (5)

Colorless substance (5.3 mg); IR (KBr): 3420, 2915, 2805, 1650, 1520, 1510, 1220 cm-1; 1H NMR (CD3OD, 300 MHz): I' 9.85 (1H, s, H-7), 7.31 (1H, d, J = 8.6 Hz, H-6), 6.57 (1H, d, J = 8.6 Hz, H-5), 5.38 (1H, d, J = 1.6 Hz, H-1'), 4.35 (1H, dq, J = 9.7 and 6.2 Hz, H5'), 4.18 (1H, dd, J = 3.3 and 1.6 Hz, H-2'), 3.95 (1H, dd, J = 9.5 and 3.4 Hz, H-3'), 3.43 (1H, t, J = 9.6 Hz, H-4'), 1.20 (3H, d, J = 6.2Hz, H-6'); 13C NMR (125 MHz, CD3OD): 196.3 (C-7), 160.0 (C-4), 157.9 (C-2), 132.5 (C-3), 132.0 (C-6), 116.6 (C-1), 109.9 (C-5), 92.2 (C-1'), 81.3 (C-2'), 73.7 (C-4'), 72.3 (C-5'), 72.0 (C-3'), 18.1 (C-6'); (+)-FABMS: m/z 323 [M+Na]+, 176 [M+Na-sugar]+; (+)-HR-FABMS: m/z 323.07394 [M+Na]+ (calcd 323.07364 for C13H16O8Na, corresponding to the formula as C13H16O8).

Hydrolysis of compound 4

To a solution of compound 4 (5 mg) in MeOH (3 ml), 2.5 ml of 1 N HCl was added, and the solution was refluxed for 4 h. The reaction mixture was concentrated under reduced pressure, diluted with 5.0 ml of distilled water, and was extracted with EtOAc (3x10 ml). The aqueous phase carried hydrozlyzed sugar, was then subjected to comparative TLC with the authentic sample of [alpha]-L-rhamnose, developved in a solvent system (EtOAc-MeOH-H2O-HOAc; 4:2:2:2).

Results and Discussion

Compound 1 was isolated as white amorphous powder, which exhibited diagnostic IR absorption bands at 3470, 3240 and 1627 cm due to hydroxyl function, secondary amine and carbonyl group respectively. ESIMS of 1 in negative mode displayed pseudo-molecular ion at m/z 180, whereas, high resolution analysis of the same ion (HR-ESIMS m/z 180.0658 [M-H-]) depicted the molecular formula C9H11NO3. The H NMR spectrum (Table) of 1 displayed three signals in the aromatic region at I' 7.62 (d, J = 1.8 Hz), 7.01 (dd, J = 8.6, 1.8 Hz) and 6.81 (d, J = 8.6 Hz), attested for a 1,3,4-trisubstituted benzene ring. An oxymethylene resonated in the same spectrum at I' 4.48 (s), which was correlated in the HSQC spectrum (Table) with a carbon resonance at I' 65.0. A 3-hydrogen singlet at I' 2.17 was correlated in HSQC spectrum with the carbon at I' 23.4, indicating an acetyl group in 1. However, the carbonyl carbon resonance at I' 172.2 in the 13C NMR spectrum suggested an ester or amide function.

The stretching absorption of the carbonyl group at 1627 cm-1 and that of secondary amine ruled out the presence of an ester group instead depicted an N-acetyl connectivity in 1. Theoretical NMR calculations of benzene ring carbons fully supported the amide function in 1 instead of an ester moiety. Various substitutions on benzene ring were fixed due to HMBC correlations (Figure 2), in which the oxymethylene (I' 4.48) was correlated with a quaternary carbon at I' 133.9 and with two aromatic methines at I' 125.8, 123.1. The respective protons of these two aromatic methines showed HMBC correlation with the oxymethylene carbon at I' 65.0, thus substantiated the position of the side chain at C-5. Further interpretation of the HMBC correlations (Figure 2) allowed placing the hydroxyl substituent at C-2 and amide function at C-1. Complete analysis and discussion of the above given data elucidated the structure 1 as N-[2-hydroxy-5-(hydroxymethyl) phenyl] acetamide, which is a new natural product.

The 1H NMR analysis of a polar fraction of the ethyl acetate extract of the culture broth exhibited mixed signal for two tri-substituted benzene rings, signal for aldehydic proton, oxymehtylene and singlet methyl. In addition, two anomeric proton resonances were observed at I' 5.43, 5.40 (d, J = 1.4 Hz each) along with other sugar methines resonances between I' 4.35-3.41. Two methyl doublets displayed their position in the same spectrum at I' 1.25 and 1.23 (J = 6.1 Hz each). This data in comparison with the 1H NMR data of compounds 1 and 6 (known compound) helped to tentatively determine the structures of compounds 2 and 3. These deductions were substantiated due to HR-FABMS analysis of the mixture, in which the ion peaks oberved at m/z 350.1214 [M+Na]+ (calcd. 350.1216 for C15H21NNaO7) and 348.1053 [M+Na]+ (calcd. 348.1059 for C15H19NNaO7). Due to low amount ( 2 mg) of the fraction and limited facilities, compounds 2 and 3 could not be separated.

However, these two metabolites have also been noticed as new natural products. The (+ve) FABMS of 4 exhibited pseudo-molecular ion peak at m/z 323 [M+Na]+, along with another fragment ion at m/z 176 [M+Na-Sugar]+ indicating the glycosidic nature of 4. The molecular formula of 4 could be determined as C13H16O8 due to (+ve)-HR-FABMS (m/z 323.0751) with six double bond equivalence (DBE). The 1H NMR spectrum (Table) of 4 displayed two resonances at I' 7.31 (1H, d, J = 8.6Hz) and 6.56 (1H, d, J = 8.6 Hz), that witnessed a tetra-substituted benzene ring. The most downfield methine singlet at I' 9.68 could be attributed to an aldehyde function, which was correlated in HSQC spectrum with a carbon signal at I' 196.4. The 1H NMR spectrum was further indicative of a rhamnosyl moiety in 4 as it exhibited the resonance of anomeric proton at I' 5.40 (d, J = 1.6 Hz), and a methyl doublet at I' 1.21 (d, J = 6.2 Hz), whose corresponding carbon appeared at I' 17.9 in the 13C NMR spectrum.

Four more oxymethines signals were also observed in the spectrum (Table). The J value (1.6 Hz) of the anomeric hydrogen depicted [alpha]-L-rhamnosyl moiety in 4, which was substantiated due to optical rotation value ([[alpha]]D20 +7.9Adeg) of the hydrolyzed product [5] and comparison of the TLC profile of separated sugar with that of the standard sample. Aldehydic proton exhibited HMBC correlation (Figure 3) with three aromatic carbons at I' 116.7 (C-1), 157.8 (C-2) and 129.9 (C-6). The data showed that compound 4 has three oxygenated aromatic carbons (I' 157.8 (C-2), 132.6 (C-3) and 159.9 (C-4). Relatively lower shift of oxygenated C-3 (132.6) revealed that it must be flanked between two other oxygenated aromatic carbon atoms (I' 157.8 and 159.9). Thus the position of sugar moiety at C-3 could be determined due to HMBC correlation of anomeric proton (I' 5.40) with the carbon at I' 132.6 (C-3).

Further analysis of the COSY and HMBC spectra along with above discussed data led to the structure of 4 as 2,4-dihydroxy-3-O-[alpha]-L-rhamnopyranosyl-benzaldehyde, which is a new natural product. The molecular formula of 5 was calculated as C14H18O8 due to a pseudo-molecular ion peak at m/z 337.0878 [M+Na]+ in the positive HR-FABMS. The molecular mass and other fragment ion at m/z 176 [M+Na-sugar]+ in the spectrum suggested 5 to be a methyl-derivative of 4. The NMR data (Table) of 5 was nearly super-imposable to that of 4 with fewer differences, that it afforded additional resonances due to a methoxyl group [I'H 3.74 (s) and I'C 59.4] along with a significant change in the carbon chemical shift (I' 81.4) of C-2' of rhamnosyl moiety. The downfield shift of this carbon could be attributed to the attachment of methoxyl group to C-2', which was substantiated due to HMBC correlation (Figure 3) of methoxyl proton (I' 3.74) with C-2' (I' 81.4).

Based on these information, compound 5 was elucidated as 2,4-dihydroxy-3-O-[2-OMe]-[alpha]-L-rhamnopyranosyl-benzaldehyde, which is also a new natural product.

Table-1: 1H (CD3OD, 300 MHz) and 13C (CD3OD, 75 MHz) NMR data of compounds 1, 4-5.

Position###1###4###5

###I'H(J in Hz)###I'C###I'H(J in Hz)###I'C###I'H(J in Hz)###I'C

###1###-###126.8###-###116.7###-###116.6

###2###-###149.0###-###157.8###-###157.9

###3###6.81(d, 8.6)###117.0###-###132.6###-###132.5

###4###7.00(dd, 8.6, 1.8)###125.8###-###159.9###-###160.0

###5###-###133.9###6.56(d, 8.6)###110.0###6.57(d, 8.6)###109.9

###6###7.62(d, 1.8)###123.1###7.31(d, 8.6)###131.9###7.31(d, 8.6)###132.0

###7###4.48(s)###65.0###9.86(s)###196.4###9.85(s)###196.3

###1###-###172.2###5.40(d, 1.6)###102.9###5.38(d, 1.6)###92.2

###2###2.17###23.4###4.17(dd, 3.3, 1.7)###72.0###4.18(dd, 3.3, 1.7)###81.3

###3###-###-###3.93(dd, 9.5, 3.4)###72.1###3.95(dd, 9.5, 3.4)###72.0

###4###-###-###3.43(t, 9.6)###73.7###3.43(t, 9.6)###73.7

###5###-###-###4.33(dq, 9.7, 6.2)###71.2###4.35(dq, 9.7, 6.2)###72.3

###6###-###-###1.21(d, 6.2)###17.9###1.20(d, 6.2)###18.1

###OMe###-###-###-###-###3.50(s)###59.4

Conclusion

3-Amino-4-hydroxybenzaldehyde (6a) is an intermediate of grixazone synthesis in Streptomyces griseus (a species of soil Actinomycetes), and aryl amine group is acetylated by cytosolic enzymes; arylamine N-acetyltransferases (NATs). [6] Although the isolate Actinomycete sp. CDS1 has not been identified fully, but due to similar nature of its secondary metabolites, i.e. acetylated derivatives of 3-amino-4-hydroxybenzaldehyde, it is speculated that the strain CDS1 may be a species of the genus Streptomyces.

It is further concluded that our isolate Actinomycete sp. CDS1 is also producing several benzaldehyde derivatives, and their reduced products benzyl-derivatives (1-3, 6), which might be being acetylated by NAT. 2,3,4-Trihydroxy-benzaldehydes are very rare in natural products, however, gallic acid or 3,4,5-trihydroxybenzaldehyde derivatives have been reported from plant sources. [7] Actinomycete sp. CDS1 is also producing 2, 3, 4-trihydroxy-benzaldehyde rhamnosides (4 and 5), which is unique characteristic to this bacterial strain.

Acknowledgments

The corresponding author is thankful to Prof. G. M. Koenig for providing scientific support in the form of equipment subsidy to her lab in Pakistan.

References

1. J. Berdy, Thoughts and facts about antibiotics: Where we are now and where we are heading; J. Antibiot., 65, 385 (2012).

2. M. S. Abdel-Salam and W. Klingmuller, Transposon Tn5 mutagenesis in Azospirillum lipoferum: isolation of indole acetic acid mutants, Mol. Gen. Genet., 210, 165 (1987).

3. B. Irlinger, A. Bartsch, H. J. Kramer, P. Mayser and W. Steglich, New tryptophan metabolites from cultures of the lipophilic yeast Malassezia furfur. Helv. Chim. Acta., 88, 1472 (2005).

4. S. K. Agadagba, Isolation of Actinomycetes from soil. J. Microbiol. Res., 4, 136 (2014).

5. M. A. Naveed, N. Riaz, M. Saleem, B. Jabeen, M.Ashraf, R. Nasar and A. Jabbar, Longipetalosides A-C, new steroidal saponins from Tribulus longipetalus. Steroids, 83, 45 (2014).

6. H. Suzuki, Y. Ohnishi, S. Horinouchi, Arylamine N-Acetyltransferase Responsible for Acetylation of 2-Aminophenols in Streptomyces griseus. J. Bacteriol. 189, 2155 (2007).

7. H. Feng, G.-I. Nonaka, I. Nishioka, Hydrolysable tannins and related compounds from Castanea mollissima Phytochemistry, 27, 1185 (1988).
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Author:Nazir, Mamona; Mustafa, Rizwana; Tousif, Muhammad Imran; Ahmad, Shabir; Khatoon, Tasneem; Ahmad, Ish
Publication:Journal of the Chemical Society of Pakistan
Article Type:Technical report
Date:Oct 31, 2018
Words:3278
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